The aim of this project is to identify and characterize factors that regulate gene expression in vertebrate embryos as an approach to understanding the mechanisms that control early development. The primary model system is an epidermal keratin gene, XK81AI, expressed in the amphibian (Xenopus laevis) embryo. The ill-regulatory elements of this gene have been mapped by introducing mutated DNA constructs into frog embryos. We have found that the Xenopus homolog of a mammalian transcription factor, AP-2 (XAP-2) binds to a specific regulatory site and stimulates the activity of the keratin promoter. We have cloned XAP-2 and are studying its interactions with the keratin gene regulatory sites. We have shown that XAP-2 RNA is localized in the epidermal and neural crest cells in early development, and in epidermis and kidney in the adult frog. We have also obtained evidence for alternatively spliced forms of XAP-2 RNA in vivo. The functional significance of these observations is being studied. The genomic XAP-2 DNA has been cloned and partially sequenced, with the aim of analyzing its regulation, using the approaches we have developed for the Xenopus keratin gene. PCR-amplified in vivo footprinting has been used to identify several additional potential regulatory sites near the keratin gene promoter, including a sequence resembling the binding sites for the mammalian transcription factor AP-1. The significance of this apparent homology is being tested.